Radiometal-binding analogues of luteinizing hormone releasing hormone

ABSTRACT

Peptide, derivatives of leutenizing hormone releasing hormone that are capable of binding radionuclides are provided. The peptide derivatives are readily labeled with isotopes of rhenium or technetium, while retaining their ability to tightly bind LHRH receptors. Methods for preparing the labeled peptides and their use in methods of radiodiagnosis and radiotherapy are described.

BACKGROUND OF THE INVENTION

This invention relates to derivatives of leutenizing hormone releasinghormones (LHRH) in which one or more of the amino acid side chainscontain chelating moieties that can tightly bind radionuclides.

Luteinizing hormone-releasing hormone (LHRH) is a decapeptide having thestructure (<G)HWSYGLRPG-NH₂, (SEQ ID NO:1) where <G is pyroglutamicacid. LHRH controls pituitary synthesis of the gonadotropins luteinizinghormone (LH) and follicle stimulating hormone (FSH). LH and FSH controlthe synthesis of sex steroids in the gonads. It has been shown thatanalogues of LHRH, when substituted in position 6, 10, or both, displayboth greater and more sustained bioactivity than native LHRH. More than3000 LHRH peptides have been evaluated both in vitro and in vivo. See,for example, Schally et al., BASIC ASPECTS; GNRH ANALOGUES IN CANCER ANDIN HUMAN REPRODUCTION, Vickery & Lunenfeld eds. Vol. 1, pp. 5-31,(Kluwer Academic Publishers, Dordecht, 1989); Schally et al., ADVANCESIN GYNECOLOGY AND OBSTETRICS, GENERAL GYNECOLOGY, Belfort et al. eds.,Vol. 6, pp. 3-20 (Parthenon Publishers, Carnforth, UK, 1989); Vickery etal., Endocrine Rev. 7: 115 (1986); Dutta et al., Drugs of the Future,13:761 (1988). Several of these analogues have been used clinically,including: [D-Leu⁶, NH-Et¹⁰] LHRH (Vilchez-Martinez et al., Biochem.Biophys. Res. Commun. 59:1226 (1974); [D-Trp⁶] LHRH (Coy et al., J. Med.Chem. 19:423 (1976); [D-Ser(tBu)⁶, NH-Et¹⁰] LHRH (Koenig et al., In:PROCEEDINGS OF THE FOURTH AMERICAN PEPTIDE SYMPOSIUM, Walter andMeienhofer eds., 883-888 (1975)); [D-Ser(tBu)⁶, NH—NH—CO—NH₂ ¹⁰] LHRH(Dutta et. al., J. Med. Chem. 21:1018 (1978); [D-Nal(2)⁶]LHRH (Nestor etal., J. Med. Chem. 25:795 (1982)).

In addition, changes in position 1, 2, 3, 6 and optionally in positions5 and 10 of the LHRH molecule can give rise to powerful antagonists. SeeKarten M. J. et al., Endocrine Review 7:44 (1986) and Bajusz, S. et al.,Int. J. Pept. Prot. Res. 32:425 (1988). These antagonists inhibit therelease of LH and FSH from the pituitary and as such, have potential asclinical agents in the imaging, diagnosis and treatment of hormonedependent cancers such as prostate, breast, ovarian, endometrial andpancreatic cancers.

The mechanism of LHRH analogue action is related, at least in part, tothe fact that the density of the LHRH receptors of human tumors may besubstantially greater than the LHRH receptor density of normal cells.Furthermore, the LHRH receptors of tumor cells possess a high affinityfor LHRH peptides. For example, 80% of epithelial ovarian cancers haveupregulated LHRH receptor densities and the receptors also have highaffinities for the LHRH peptides. See Emons et al., Cancer Res. 53:5439(1993); Irmer et al., Cancer Res. 55:817 (1955). Similarly, LHRHreceptors have also been shown to be upregulated in breast cancer tumors(Fekete et al., Endocrinol. 124:946 (1989); Fekete et al., J. Clin. Lab.Anal. 3:137 (1989), endometrial cancers (Srkalovic et al., Cancer Res.50:1841 (1990)), prostate tumors (Srkalovic et al., Endocrinol. 127:3052(1990)), and pancreatic cancers (Schally et al., J. Steroid Biochem.Molec. Biol. 37: 1061 (1990)).

It has been shown that analogues of LHRH will selectively bind tohormone-sensitive tumors which are characterized by an overexpression ofhormone receptors on the cell surface. When LHRH responsive tumors aretreated with LHRH peptide analogues the analogues bind to the receptorson the cell surface and are then internalized. See Jackson et al.,Cancer Treat. Rev. 16:161 (1989). Some studies have been carried out inwhich LHRH agonist and antagonist derivatives containing cytotoxicmoieties attached to the targeting LHRH peptide have been used todeliver the cytotoxin into the cell. LHRH analogues modified withspecific cytotoxic moieties may, therefore, be useful as carriers forchemotherapeutic agents. See, for example, EP 0 450 461 A2 and EP 0 364819 A2. It has further been shown that, provided the analogues arelipophilic, various substituents can be attached to the side chain ofthe amino acid at position 6 of LHRH while still retaining its activityboth in vitro and in vivo. (Janaky, T. et al. Proc. Natl. Acad. Sci. USA89:972 (1992). Cytotoxic metal complexes containing platinum, nickel,and copper attached to the side chain of lysine at position 6 havedemonstrated high in vitro activity in human breast tumor cells. SeeBajusz, S. et al. Proc. Natl. Acad. Sci. USA 86:6313 (1989).

Some peptides either directly possesses, or are amenable to theintroduction of residues that allow direct binding of radiometals to thepeptide. For example, somatostatin contains a disulfide bond that, uponreduction, provides two sulfhydryl-containing cysteine side chains thatcan directly bind ^(99m)Tc. See U.S. Pat. No. 5,225,180. See also WO94/28942, WO 93/21962 and WO 94/23758. Complexes of this type tend,however, to be heterogeneous and unstable and, moreover, the use of freesulfhydryls in this manner limits the radiometals which can be used tolabel the peptide to those that tightly bind free S—H groups. Thismethods also suffers from the problem that direct binding of the metalto an amino acid side chain can greatly influence the peptideconformation, thereby deleteriously altering the receptor bindingproperties of the compound.

Alternatively, chelating agents can be introduced into peptide sidechains by means of site-selective reactions involving particular aminoacid residues. For example, the lysine residue at position 6 of LHRH hasbeen directly acylated with a chelating group. Bajusz et al. supra. Thismethod is inherently limited by the lack of selectivity available whenmore than one side chain can potentially react with the chelator, orwhen the peptide sequence does not contain an amino acid that can bederivatized in this way.

Most peptides either do not contain a metal-binding sequence motif or,for various reasons such as those described supra, are not amenable tosuitable sequence modifications that would permit introduction of such amotif. Some means of rendering the peptide capable of bindingradiometals must therefore be introduced into the peptide. A preferredapproach is to attach a metal binding ligand to the peptide so that asingle, stable complex is formed. The ligands used to bind metals oftencontain a variety of heteroatoms such as nitrogen, sulfur, phosphorous,and oxygen that have a high affinity for metals.

These ligands are typically attached at the N-terminus of the desiredpeptide. This allows the peptide chain to be constructed usingconventional methods of peptide synthesis, followed by addition of theligand once peptide synthesis is complete. For example, Maina et al.have described the coupling of a tetra-amine chelator to the N-terminusof a somatostatin analogue, which then allowed ^(99m)Tc labeling of thepeptide. See J. Nucl. Biol. Med. 38:452 (1994). Once again, however,application of this method is limited to those circumstances in whichthe N-terminus of the peptide can accommodate the presence of a (usuallybulky) chelator without deleteriously affecting the binding propertiesof the peptide.

Bajusz et al., supra also describe the incorporation of a protected,chelate-derivatized lysine residue into a growing peptide chain duringpeptide synthesis. This method, however, requires the preparation of asuitably derivatized lysine derivative that also bears an α-aminoprotecting group that is compatible with peptide synthesis. It wouldclearly be preferable to be able to use protected amino acidsderivatives that are commercially available for use in peptidesynthesis, and to subsequently deprotect and derivatize appropriateamino acid side chains in a selective fashion.

It is apparent, therefore, that a means of attaching a chelating moietyto any predetermined position within a peptide is greatly to be desired.It is also desirable to have access to a method that would allow thischelating moiety to be coupled to the peptide at any desired stageduring peptide synthesis.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide analoguesof LHRH that can bind radionuclides while retaining the ability tospecifically bind to the LHRH receptor. It is a further object of theinvention to provide methods of preparing and radiolabeling analogues ofLHRH that can bind radionuclides while retaining the ability tospecifically bind to the LHRH receptor. It is a still further object ofthe invention provide diagnostic and therapeutic methods of using theradiolabeled analogues of LHRH to image or treat a tumor, an infectiouslesion, a myocardial infarction, a clot, artherosclerotic plaque, or anormal organ or tissue, peptides.

These and other objects of the invention are achieved, inter alia, byproviding a peptide comprising the amino acid sequenceX¹-X²-X³-ser-X⁴-X⁵-X⁶-X⁷-pro-X⁸-NH₂, (SEQ ID NO:2) where X¹ ispyroglutamic acid or D-acftylnaphthylalanine, X² is histidine orD-4-chlorophenylalanine, X³ is D- or L-tryptophan or tyrosine, X⁴ istyrosine, leucine, or arginine, X⁵ is a D- or L-amino acid derivativecapable of chelating a radiometal, X⁶ is leucine or tryptophan, X⁷ isarginine or lysine, and X⁸-NH₂ is glycine amide or D-alanine amide.

In accordance with one aspect of the invention X⁵ is:

where R¹ is H, OH, a peptide, a sugar, a targeting molecule, loweralkyl, substituted lower alkyl, or a protecting group that can beremoved under the conditions of peptide synthesis; R² is H, lower alkyl,or substituted lower alkyl; W is from 1-20 atoms long and is selectedfrom the group consisting of cycloalkyl, aryl, or alkaryl groups, asubstituted or unsubstituted alkylene chain, and a chain substitutedwith at least one heteroatom; Z is a peptide containing 1-5 residues, orZ is COCH₂ or COCH(CH₂SP²), in which p² is H or a sulfur protectinggroup; and A and D are the same or different, and each is selected fromthe group consisting of H, COCH₂NR³NR⁴C(S)NHR⁵, COCH₂NR⁶NR⁷C(S) NHR⁸,COCH₂NR⁹NR¹⁰C(O)CH₂SP², CONR¹¹NR¹²C(O) CH₂SP², NR¹³C(S)NHR¹⁴, orCOCH₂NR¹⁵COCH₂SP², R³, R⁴, R⁶, R⁷, R⁹, R¹⁰, R^(11,R12), R¹³, and R¹⁵ arethe same or different, and each represents H, lower alkyl, orsubstituted lower alkyl, and R⁵, R⁸, and R¹⁴ are the same or differentand each is H, lower alkyl, substituted lower alkyl, aryl, orsubstituted aryl.

In a preferred embodiment of the invention X⁵ is selected from the groupconsisting of:

In accordance with another aspect of the invention there are providedpeptides in which X¹ is pyroglutamic acid, X² is histidine, and X⁵ isglycine amide. In preferred embodiments of this aspect of the inventionX³ is tyrosine, X⁴ is leucine, X⁶ is tryptophan, and X⁷ is lysine. Inother preferred embodiments of this aspect of the invention X⁵ is

In other preferred embodiments of this aspect of the invention, X³ istryptophan, X⁴ is tyrosine, X⁶ is leucine, X⁷ is arginine, and X⁵ is

In accordance with still another aspect of the invention there isprovided peptides in which X¹ is D-acetylnaphthylalanine, X² isD-4-chlorophenylalanine, X³ is D-tryptophan, X⁴ is arginine, X⁶ isleucine, X⁷ is arginine, and X⁸—NH₂ is D-alanine amide. In preferredembodiments X⁵ is

In accordance with yet another aspect of the invention there areprovided peptides in which X¹ is D-acetylnaphthylalanine, X² isD-4-chlorophenylanine, X³ is D-tryptophan, X⁴ is arginine, X⁶ istryptophan, X⁷ is lysine, and X⁸—NH₂ is glycine amide.

In accordance with a yet further aspect of the invention there isprovided a method of preparing a metal-chelating composition, comprisingcontacting a solution of a peptide with stannous ions, where the has theamino acid sequence described above, and then contacting this solutionwith a radionuclide and recovering the radiolabeled peptide. In apreferred embodiment the radionuclide is selected from ¹⁸⁸Re- or¹⁸⁶-perrhenate and ⁹⁹Tc-pertechnetate.

In accordance with yet another aspect of the invention there areprovided peptides that specifically bind cells or tissues that expressLHRH receptors.

In accordance with another aspect of the invention there is provided amethod of imaging a tumor, an infectious lesion, a myocardialinfarction, a clot, artherosclerotic plaque, or a normal organ ortissue, comprising administering to a human patient a radiolabeledpeptide that specifically binds to cells or tissues that express LHRHreceptors, together with a pharmaceutically acceptable carrier, and,after a sufficient time for the radiolabeled peptide to localize and fornon-target background to clear, the site or sites of accretion of theradiolabeled peptide are detected by an external imaging camera, whereinthe radiolabeled peptide is prepared by the method described above.

DETAILED DESCRIPTION

The present invention provides analogues of leutenizing hormonereleasing hormone (LHRH) that are capable of binding radionuclides.These analogues are prepared by site-specifically introducingradionuclide-chelating amino acid derivatives into peptides that aresynthesized by solid-phase or solution phase methods.

The synthesis of the analogues involves the use of differentiallyprotected bis-amino acid derivatives in which either amino function canbe selectively deprotected. These derivatives are introduced into agrowing peptide chain during peptide synthesis by conventional peptidecoupling methodology. One of the amino functions is then selectivelydeprotected, allowing subsequent coupling of either a chelatingmolecule, or addition of further amino acid residues to continue thepeptide synthesis.

If peptide synthesis is continued, selective deprotection of the secondamino group of the bis-amino acid can be accomplished at any pointduring the peptide synthesis to introduce the chelating moiety. Once thepeptide synthesis is complete, cleavage, deprotection, and purificationaffords the peptide derivative. This derivative is then labeled with aradiometal for use in radiodiagnostic and radiotherapeutic applications.

Alternatively, if the chelating molecule is coupled to the deprotectedamino group first, the second step is to deprotect the other amino groupand continue with the peptide synthesis. Final cleavage, deprotectionand purification steps yield the pure peptide derivative, which is thenradiolabeled as before.

The radiometal chelating peptides of the present invention are stable inblood and other bodily fluids and tissues. Both the reagents and theconditions in the present method are greatly simplified over those inthe prior art, and the labeled peptides are particularly suitable forradiodiagnostic and radiotherapy applications using technetium orrhenium labeling.

The approach outlined above allows the placement of a radiometal-bindingamino acid anywhere in the LHRH peptide sequence. Placing the chelatingmoiety on an amino acid side-chain, rather than the N-terminus of apeptide, has the added advantage of spatially distancing the metalcomplex from the peptide backbone, thereby minimizing the effect of themetal complex on the peptide conformation.

It is known that peptide conformation is greatly influenced by chargeand hydrophilic/hydrophobic interactions, and it is therefore importantto consider these variables when designing a chelating ligand to be usedin peptides. It is preferred that a variety of chelating complexes ofvarying charge and hydrophilicity are prepared and tested to select themetal-complexed LHRH peptide that displays the optimum combination oftarget selectivity and chelate stability.

The radiolabeled LHRH peptides of the present invention bindspecifically to a diseased cell or tissue that exhibits both a high LHRHreceptor density and high affinity for LHRH. The radioactivity of theradionuclide allows diagnosis and/or treatment of the tumor or diseasedtissue. The invention also includes pharmaceutical compositionscomprising an effective amount of at least one of the radiolabeledpeptides of the invention, in combination with a pharmaceuticallyacceptable sterile vehicle, as described, for example, in Remington'sPharmaceutical Sciences; Drug Receptors and Receptor Theory, 18th ed.,Mack Publishing Co., Easton, Pa. (1990). The invention also includeskits for labeling peptides which are convenient and easy to use in aclinical environment.

Design and Synthesis of Peptides Incorporating Chelating Amino AcidDerivatives

The peptides of the invention contain radiometal-chelating amino acidderivatives that are characterized by the presence of at least one thiolor thiocarbonyl group, and at least one nitrogen present as either atertiary amine or a secondary amide. The sulfur and nitrogen atoms aresuitably disposed to form a multidentate ligand capable of tightly andpreferentially binding reduced radionuclide. These amino acidderivatives are incorporated into peptides that bind tightly to the LHRHreceptor. These peptides can be represented generally by the formula:

X¹-X²-X³-ser-X⁴-X⁵-X⁶-X⁷-pro-X⁸-NH² (SEQ ID NO:2)

where

X¹ is pyroglutamic acid or D-acetylnaphthylalanine;

X² is histidine or D-4-chlorophenylalanine;

X³ is D- or L-tryptophan or tyrosine,

X⁴ is tyrosine, leucine, or arginine,

X⁵ is a radiometal-chelating amino acid as described below;

X⁶ is leucine or tryptophan;

X⁷ is arginine or lysine; and

X⁸—NH₂ is glycine amide or D-alanine amide.

The radiometal-chelating amino acid derivatives contemplated by theinvention can be represented by the general formula:

is which R¹ represents H, OH, a peptide, a sugar, a targeting molecule,lower alkyl, substituted lower alkyl, or a protecting group that can beremoved under the conditions of peptide synthesis, R² is H, lower alkyl,or substituted lower alkyl, W is from 1-20 atoms long and is selectedfrom the group consisting of cycloalkyl, aryl, alkaryl, a substituted orunsubstituted alkylene chain, and a chain substituted with at least oneheteroatom, Z is a peptide containing 1-5 residues, or Z is COCH₂ orCOCH(CH₂SP²), in which P² is H or a sulfur protecting group, A and D arethe same or different, and each is selected from the group consisting ofH, COCH₂NR⁴C(S)NHR⁵, COCH₂NR⁶NR⁷C(S)NHR⁸, COCH₂NR⁹NR¹⁰C(O)CH₂SP²,CONR¹¹NR¹²C(O) CH₂SP², NR¹³C(S)NHR¹⁴, or COCH₂NR¹⁵COCH₂SP², R³, R⁴, R⁶,R⁷, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁵ are the same or different, and eachrepresents H, lower alkyl, or substituted lower alkyl, and R⁵, R⁸, andR¹⁴ are the same or different and each is H, lower alkyl, substitutedlower alkyl, aryl, or substituted aryl.

Representative embodiments of radiometal-chelating amino acidderivatives of the invention are:

Each of the chelating amino acids of the invention can be prepared bymethods well known to the skilled practitioner in the art of organicsynthesis. Detailed protocols for the synthesis of representativechelates are given in the examples found below.

The chelating amino acids are constructed from subunits that are linkedtogether by simple coupling or condensation reactions, such as thecondensation of an amino, hydrazino, or hydrazido function with anactivated carboxyl group, or reductive amination reactions betweenamines and aldehydes. As used herein the term “condensation” is intendedto encompass reactions that couple together subunits of the chelatingmoiety, and thus encompasses reactions such as reductive amination inaddition to reactions that conform to the classical definition of acondensation reaction.

Following a condensation reaction, additional functional groups on thesubunit may be deprotected to allow additional condensation reactions.For example, a second subunit carrying a free carboxyl group and aprotected amino function can be condensed with an amino, hydrazino, orhydrazido function on a first subunit. The amino function on the secondsubunit moiety can then be deprotected and further coupled to a thirdsubunit.

Methods of activating carboxyl groups for such condensation reactionsare well known to those of skill in the art of organic synthesis andpeptide synthesis, and include the use of active esters and ofcarbodiimide coupling agents. Suitable protecting groups are used forprotecting functions on the subunits when the reactivity of thefunctions is incompatible with a reaction used to join the subunits.Protecting groups for both amino and carboxylic acid functions are wellknown in the art. See, for example, Greene, supra. The subunits used toconstruct the chelate are either readily prepared by method well knownin the art, or are commercially available from suppliers such asAdvanced ChemTech (Lexington, Ky.), Milligen (Burlington, Mass.),Applied Biosystems (Foster City, Calif.), or Aldrich Chemical Corp.(Milwaukee, Wis.).

The condensation reactions used to link together the subunits can eitherbe carried out prior to peptide synthesis, or during the peptidesynthesis process. When the amino acid derivative is assembled from itssubunits prior to peptide synthesis, α-amino and α-carboxyl functionsmust be suitably protected in a manner that is subsequently compatiblewith selective deprotection and activation of these functionalities forpeptide synthesis. Examples of such protecting groups are well known inthe art, and include the fluorenemethyloxycarbonyl (Fmoc),benzyloxycarbonyl (Cbz), ^(t)butoxycarbonyl (Boc), and allyloxycarbonyl(alloc) groups for amino protection. Groups for carboxyl protectioninclude the methyl (Me), benzyl (Bn), ^(t)butyl (^(t)Bu), and allylesters, respectively. The amino and carboxyl protecting groups must beselected such that each group can be selectively deprotected in thepresence of the other. This precludes, for example, use of the Cbz groupfor protection of the amino function in the presence of a carboxyl groupprotected as a benzyl ester. See Greene, supra. In a preferredembodiment, the α-amino group is protected as an Fmoc group, and theα-carboxyl group is a methyl ester. The thiol protecting group used inthe compounds of the invention can be any organic or inorganic groupwhich is readily removed under mild conditions to regenerate the freesulfhydryl in the presence of the protein. Suitable protecting groupsare listed in Greene, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (WileyInterscience, NY. 1981) pp. 193-217. Examples of suitable protectinggroups include trityl groups, thiol esters, thiocarbamates anddisulfides. In a preferred embodiment the thiol protecting group is atrityl group. Those skilled in the art are familiar with the proceduresof protecting and deprotecting thiol groups. For example, benzoatethioesters may be deprotected under mild and selective conditions usinghydroxylamine.

Once assembly of the protected chelating moiety is complete, theα-carboxy function is deprotected and coupled to the amino terminus ofthe peptide chain using conventional methods of peptide synthesis. SeeBodanszky et al., THE PRACTICE OF PEPTIDE SYNTHESIS (Springer Verlag,Heidelberg, 1984).

When the amino acid derivative is assembled from its subunits duringpeptide synthesis, the peptide chain is assembled by conventional solidphase synthesis until the point where the derivative is to beincorporated. The differentially protected bis-amino acid is thencoupled to the amino terminus of the peptide chain, followed byselective deprotection of one of the amino groups of the derivative.

If the α-amino function is deprotected first, all or part of theremaining amino acid residues are then coupled to the peptide chain inthe conventional manner. The side chain amino function of the derivativeis then deprotected, and the chelating moiety is assembled as describedabove. The complete peptide can then be deprotected and purified bystandard methods.

If the side chain amino function is deprotected first, the chelatingmoiety is then assembled as described above, followed by deprotection ofthe α-amino group. Peptide synthesis is then completed in theconventional manner as described above.

Once peptide synthesis is complete the fully protected peptide isdeprotected and purified. Methods for deprotection and purification ofsynthetic peptides are well known in the art. See, for example,Bodanszky, supra. If the peptide was synthesized by solid phasetechniques the peptide must also be cleaved from the resin used as thesolid support for the synthesis. Methods for achieving this cleavage arealso well known in the art. Methods for purifying synthetic peptidessuch as those of the present invention are also well known to those ofskill in the art. Such methods include, for example, ion exchange, gelfiltration chromatography, and reversed phase high pressure liquidchromatography (RP-HPLC). In a preferred embodiment of the invention thepeptide is purified by RP-HPLC using a preparative scale octadecylsilane(C18) silica column packing, eluting with a gradient of acetonitrile in0.1% trifluoroacetic acid (TFA). The purity of the peptide can beconfirmed by standard methods such as analytical RP-HPLC or capillaryelectrophoresis. The identity of the peptide can be confirmed by NMRspectroscopy, or in a preferred embodiment of the invention, by massspectrometry.

Chelation of Radiometals by Peptides Incorporating Metal-Chelating AminoAcid Derivatives

Once a peptide incorporating a metal-chelating amino acid derivative hasbeen synthesized and purified it can be stored for later use, or it canbe reacted with radionuclide for immediate use in radioimmunotherapy orradioimmunodiagnostic procedures. If the peptide is to be stored forlater use, the free thiol groups are preferably protected againstoxidation. In a preferred embodiment of the invention this can beachieved by storing the peptide under an inert atmosphere, oralternatively the peptide can be stored in the presence of a reducingagent such as β-mercaptoethanol. Storage of the peptide in a formbearing free sulfhydryl groups can also be achieved by admixing theconjugate with the agent to be used for reducing the radionuclide. Forexample, the added reducing agent is a tin^(II) salt. The salt can begenerated as required from tin metal, e.g., foil, granules, powder,turnings and the like, by contact with aqueous acid, e.g., Hcl and isusually added to the peptide in the form of SnCl₂, advantageously in asolution that is about 0.1 mM in HCl. The resulting mixture can bestored as a frozen solution, or preferably is stored as a lyophilizedpowder. Storage of the conjugate in the presence of a reducing agent inthis form is advantageous because it not only prevents reoxidation ofthe thiol functions, but also dispenses with the requirement of anadditional step to reduce the radionuclide, as discussed below.

The peptide or peptide-reducing agent mixture can be assembled into asingle vial or kit for use in performing the radiolabeling method of thepresent invention. A radionuclide then can be added to the kit as neededto provide a radiolabeled peptide. The single vials or kits of thepresent invention are designed to contain the appropriate peptide forany particular diagnostic or therapeutic procedure.

In accordance with the present method, the vials or kits advantageouslyare sealed and provided with a mechanism of introducing or withdrawingreagents under sterile or semi-sterile conditions. Preferably, a vialcontaining a port for syringe injection is used in the present method.The reagents in the vials or kits typically are provided in aqueous,frozen or lyophilized form. In one embodiment the reagents can be storedat low temperature, e.g., in the refrigerator, for several days toseveral weeks, preferably at a pH of about 3.5-5.5, more preferably atpH 4.5-5.0, advantageously under an inert gas atmosphere, e.g., nitrogenor argon.

It also is within the scope of the present invention to provide thereagents in lyophilized form for ease of storage and stabilization. Thisis advantageously effected at a pH of about 5.5, from a solution of avolatile buffer, e.g., ammonium acetate, and preferably also in thepresence of a stabilizer to prevent aggregation, e.g., a sugar such astrehalose or sucrose. Such lyophilization conditions are conventionaland well known to the ordinarily skilled artisan.

The labeling procedure of the present invention then can be performedsimply by adding the radioisotope directly from the generator e.g., inthe form of aqueous sodium pertechnetate, to the peptide or the reducingagent-chelating peptide mixture. The contents of the vial then are mixedand incubated for time sufficient to effect labeling of the peptide. Theduration and condition of incubation are not crucial, but incubationtypically is carried out for a period of time sufficient to obtainsubstantially 100% binding of radioisotope to the protein. As notedabove, different radionuclides require more or less extensive reducingconditions, and thus the length of the incubation will also depend onthe identity of the radionuclide used. “Substantially 100% binding”denotes greater than 98% radionuclide incorporation, advantageously,greater than 99% and more advantageously 100% incorporation. Usually,the incubation is conducted for a period of time of from about 0.1 toabout 60 minutes, but in a preferred embodiment is conducted for about 1to about 5 minutes. The radiolabeled peptide then can be withdrawn fromthe vial, and immediately used since further separation or purificationis not required.

In a preferred embodiment of the invention the labeling of the peptidechelate is carried out by mixing the peptide with aradiometal-glucoheptonate complex to effect transchelation from theglucoheptonate to the peptide. This method is particularly preferredwhen the radiometal is technetium-99. This procedure is convenientlycarried out using a Glucoscan kit (E. I. DuPont de Nemours, Inc. Boston,Mass.). The labeling is preferably carried out at room temperature insaline solution. If the peptide is not very soluble in saline asolubilizing agent such as ethanol or 2-hydroxypropyl-b-cyclodextrin maybe added. The labeling may also be carried out at elevated temperatures,such as 50°-100° C., in order to increase the rate of the labelingreaction. Protocols for labeling peptides of the invention withtechnetium and rhenium are illustrated further in Examples 9 and 10infra.

Pertechnetate for labeling peptides with ^(99m)Tc generally is obtainedfrom a commercially available generator, most commonly in the form ofNaTcO₄ in a saline solution. Other forms of pertechnetate may be used,with appropriate modification of the procedure, as would be suggested bythe supplier of a new form of generator or as would be apparent to theordinarily skilled practitioner. Pertechnetate is generally used at anactivity of about 0.2-10 mCi/ml in saline, e.g., 0.9% (“physiological”)saline, buffered at a pH of about 3-10, preferably at about 4.5-9.0.Suitable buffers include, e.g., acetate, tartrate, citrate, phosphateand the like.

Throughout this description, the phrases “reduced pertechnetate” or“reduced perrhenate” denote the species of technetium or rhenium ionformed by reduction of pertechnetate or perrhenate with, for example,stannous ion, and chelated by the thiol group(s). It is generallythought that reduced pertechnetate is in the form of Tc(III) and/orTc(IV) and/or Tc(V) in such chelates, and that reduced perrhenate is inthe form of Re(III) and/or Re(IV) and/or Re(V), but higher or loweroxidation states and/or multiple oxidation states are included withinthe scope of the present invention.

Rhenium is found just below technetium in the periodic table, has thesame outer shell electronic configuration and therefore is expected tohave very similar chemical properties, especially with respect to itsbehavior with analogous compounds. The skilled practitioner is capableof modifying the present invention based on the disclosure of technetiumlabeling to achieve efficient rhenium labeling.

The radioisotope Rc-186 is attractive for radioimmunotherapy and canalso be used for imaging. Re-188 is a generator-produced beta and gammaemitter with a half-life of about 17 hours and is suitable for imagingand therapy. Complexation of the peptides of the invention with rheniumis carried out in essentially the same manner as is described fortechnetium, supra.

For preliminary studies such as measurement of affinity constants, invitro screening, etc. for metal-bound peptides, non-radioactive rheniumis conveniently used. This allows the properties of the peptide-rheniumcomplexes to be studied without the risks associated with the handlingof radioactive rhenium. Use of non-radioactive rhenium also acts as aconvenient model for the behavior of technetium complexes of thepeptides, since no non-radioactive isotope of technetium exists, and thechemical properties of rhenium and technetium are very similar.

Once the peptide derivative has been radiolabeled it is important toconfirm that the radiolabeled conjugate retains the receptor bindingspecificity of native LHRH. Methods for determining the activity of LHRHanalogues are well known in the art. For example, a competitive cellbinding assay can be used. Target cells, for example human breastadenocarcinoma cell lines MCF-7, SK-BR-3, and MDA-MB-231 (American TypeCulture Collection, Rockville, Md.) are used in a standard assay formatin which cells are treated with different concentrations of the labeledor unlabeled peptides of the invention in the presence of LHRH (AmershamLife Science, Arlington Heights, Ill.). The radioactivity associatedwith the cells is counted and the concentration of the unlabeled LHRHthat causes 50% inhibition of the binding of the labeled LHRH analoguesis determined. The equilibrium association constant, K_(a), and thetotal number of receptor sites per cell may be determined by Scatchardanalysis. See Fersht, ENZYME STRUCTURE AND MECHANISM, 2d ed. (W. H.Freeman, London, 1985).

The ability of the radiolabeled peptide to retain radiolabel inphysiological solution can be measured using techniques essentiallysimilar to those used for radiolabeled antibodies. See Hnatowich et al.,J. Nucl. Med. 34:109 (1993). For example, assays can be used todetermine the ability of the peptide to retain radiolabel in saline andserum solution, and in the presence or absence of material such as humanserum albumin, DTPA, DOTA, cysteine and glutathione.

The in vivo bioactivity of the radiolabeled peptides of the invention isreadily determined by standard biodistribution studies in animal models,using for example, MCF-7 tumor cells grown in estrogen-dosed nude mice.In these studies it is useful to determine the receptor capacity of micebearing LHRH-binding tumors in order to estimate the quantities ofradiolabeled peptide required for imaging and/or therapy experiments.For this purpose, carrier free ¹²⁵I LHRH (˜2000 Ci/mmol, Amersham LifeScience) is injected into mice bearing MCF-7 tumors, and the mice aresacrificed at defined time intervals post-injection. The major organs,as well as the blood, and the tumor are removed, weighed, and counted todetermine the percent injected dose per gram (% ID/g) in each organ.Increasing amounts of unlabeled LHRH are then mixed with the ¹²⁵I LHRHand injected into the tumor-bearing nude mice, which are sacrificed asabove, at the same time points determined from the previous experiment.This allows the determination of the LHRH receptor capacity in the nudemouse model.

Radiolabeled peptides that show in vitro receptor affinity as determinedabove are then screened in the MCF-7 nude mouse model. The Tc-99mlabeled peptide can be purified by HPLC to obtain the peptide carrierfree metal complex for these studies if the LHRH receptor capacity istoo low to tolerate the presence of excess peptide. The biodistributionof the radiolabel is monitored on, for example, for ^(99m)Tc-labeledpeptides, a gamma camera equipped with a pinhole collimator. In theinitial screen the animals will be sacrificed after 4 hr and thebiodistribution determined as described above. Peptides that displaytumor uptake and a significant tumor to nontarget profile are thentested in a blocking assay using LHRH to determine of the tumor uptakein vivo is specific. The tumor to nontarget profile of a radiolabeledpeptide of the invention is significant if the size and location of thetumor can be determined under standard imaging conditions using thepeptide.

E. Administration of the radiolabeled peptide for diagnosis and therapy.

The peptides of the invention may be used for diagnosis or therapy ofany physiological condition in which cells or tissue express highnumbers of LHRH receptors, or express LHRH receptors of high affinity,or both. The peptides may advantageously be stored in kits as describedabove. These may be frozen or lyophilized in sterile containers under aninert gas atmosphere, and are advantageously gently thawed just prior touse. The kits are conveniently supplemented with sterile vials ofbuffers, saline, syringes, filters and other auxiliaries to facilitatepreparation of injectable preparations ready for use by the clinician ortechnician. The clinician or technician can then conveniently add asolution of a suitable radionuclide just prior to administration to apatient.

Generally, the dosage of administered labeled peptide will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition, and previous medical history. Typically, itis desirable to provide the recipient with a dosage of protein which isin the range of from about 1 pg/kg to 10 μmg/kg (amount of agent/bodyweight of patient), although a lower or higher dosage may also beadministered.

For therapeutic applications, about 0.1-500 micrograms of radiolabeledpeptide will be administered, normally daily for a period of severaldays. Administration of radiolabeled peptides to a patient can beintraveneous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. Administration byinjection may be by continuous infusion, or by single or multipleboluses.

The radiolabeled peptides of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby they are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990).

For purposes of radiotherapy, a radiolabeled peptide and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a radiolabeledpeptide and a pharmaceutically acceptable carrier is said to beadministered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient.

Additional pharmaceutical methods may be employed to control theduration of action of a radiolabeled peptide in a therapeuticapplication. Control release preparations can be prepared through theuse of polymers to complex or adsorb the protein. For example,biocompatible polymers include matrices of poly(ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebacic acid. Sherwood et al., Bio/Technology 10:1446-1449(1992). The rate of release of a peptide from such a matrix depends uponthe molecular weight of the peptide, the amount of peptide within thematrix, and the size of dispersed particles. Saltzman et al.,Biophysical. J. 55:163-171 (1989); and Sherwood et al., supra. Othersolid dosage forms are described in REMINGTON'S PHARMACEUTICAL SCIENCES,18th Ed. (1990).

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1

Synthesis of N^(α)Alloc-Ne-Fmoc-L-Lycine

N^(e)-Fmoc-L-Lysine (10.00 g, 27.1 mmol, 100 mol %, Bachem Biosciences,Inc.) was suspended in dioxane (100 ml) and Na₂CO₃ (1M, 33 ml) to form amilky suspension. Allyl chloroformate (3.2 ml, 30.2 mmol, 111 mol %) wasadded to dioxane (10 ml) and this solution was added dropwise to thesuspension of N^(e)-Fmoc-L-Lysine over 10 min. Sodium carbonate, (1M, 20ml) was added in two portions and an additional quantity of allylchloroformate (0.3 ml) was added. The reaction was stirred at roomtemperature for 16 hours. The volatile solvents were removed underreduced pressure and the residue was washed with diethyl ether (50 ml).The residual liquid was then acidified with HCl (1M) and extracted withethyl acetate (2×150 ml). The organic layers were combined, washed withsaturated NaCl (50 ml), dried over Na₂SO₄, evaporated under reducedpressure to obtain a crude oily product (16 g). The crude product wasdissolved in ether (100 ml) and a white solid formed and was removed byfiltration. The solvent from the filtrate was removed under reducedpressure to afford a viscous pale yellow oil (8.34 g, 68% yield) whicheventually formed a glassy solid.

Example 2

Synthesis of 2-(triphenylmethylmercapto)acetyl hydrazide

2-(triphenylmethylmercapto) acetic acid (20.35 g, 60.9 mmol, 100 mol %)was dissolved in anhydrous THF (150 ml) and cooled in an ice water bath.t-Butylcarbazate (8.61 g, 65.1 mmol, 107 mol %) was added to thereaction solution followed by diisopropylcarbodiimide (10.0 ml, 63.9mmol, 105 mol %). The reaction was allowed to warm slowly to roomtemperature and stirred for 28 hours. The reaction mixture was filteredto remove the white precipitate that had formed and the filtrate wasconcentrated to a white foam by removal of the solvent under reducedpressure. This material was dissolved in chloroform (75 ml). Then aceticacid (75 ml) was added followed by the addition of borontrifluorideetherate (10.0 ml, 81 mmol, 134 mol %). The reaction was stirred at roomtemperature for 6 hours and then quenched by pouring the reactionmixture into water (200 ml) containing sodium acetate (30 g). Thismixture was extracted with chloroform (2×100 ml). The organic layerswere combined, washed with saturated NaCl solution (150 ml), dried overNa₂SO₄ and filtered. The solvent was removed under reduced pressure toobtain a pale gold oil which solidified on standing. The solid wassuspended in 1:1 diethylether/hexanes (200 ml) and collected byfiltration. The solid was washed with an additional quantity of 1:1diethylether/hexanes (100 ml) and dried to afford the desired product(15.44 g, 73% yield) having ESMS MH+calculated 349, observed 349.

Example 3

Synthesis of N^(β)-[2-(triphenylmethylthio) acetyl]azaglycine

Glyoxylic acid monohydrate (0.59 g, 6.41 mmol, 110 mol %) was dissolvedin methanol (20 ml) and 2-(triphenylmethylmercapto)acetyl hydrazide(2.03 g, 5.82 mmol, 100 mol %) was added. Dioxane (20 ml) was added tothe cloudy reaction mixture and the reaction was stirred at roomtemperature for 18 hours. Sodium borohydride (1.76 g) was added to thereaction mixture and after 30 minutes, another quantity of sodiumborohydride (0.60 g) was added. The reaction was stirred for 3 hours atroom temperature, then quenched by pouring the reaction mixture into HCl(1M, 60 ml). The mixture was extracted with ethyl acetate (2×50 ml). Theorganic layers were combined, washed with saturated NaCl solution (40ml), dried over Na₂SO₄, filtered, and concentrated under reducedpressure on the rotary evaporator to afford a solid (2.5 g) having ESMSMH⁺ calculated 407, found 407.

Example 4

Synthesis of N^(Δ)-Boc-Nβ-[2-(triphenylmethylthio)acetyl]azaglycine

N^(β)-[2-(triphenylmethylthio)acetyl]azaglycine (2.39 g, 5.89 mmol, 100mol %) was dissolved in dioxane (50 ml). Di-t-butyl dicarbonate (BOC)₂O,(2.07 g, 9.48 mmol, 161 mol %) was added to the reaction solutionfollowed by the addition of Na₂CO₃ (1M, 15 ml). This mixture was stirredat room temperature for 15 minutes, then additional quantities of Na₂CO₃(1M, 10 ml) and (BOC)₂O (1.41 g) were added. The solution was stirred atroom temperature for 18 hours then reacted with NaOH (6M, 3 ml) and(BOC)₂O (1.4 g) for 1 hour. The crude reaction mixture was thenacidified to pH 3 with citric acid (1M) and extracted with ethyl acetate(200 ml). The organic layer was washed with saturated sodium chloridesolution (60 ml), dried over Na₂SO₄, filtered and concentrated underreduced pressure to obtain the crude product. The crude product wasdissolved in ether and diluted to obtain a 1:1 mixture with hexanescausing a white precipitate to form. The white solid was collected byfiltration to obtain the desired product (1.48 g, 50% yield) having ESMSMH⁺ calculated 507, found 507.

Example 5

Synthesis of 2-(4-Phenyl-3-thiosemicarbazidyl)acetic acid

4-Phenyl-3-thiosemicarbazide (6.02 g, 36 mmol, 100 mol %) was suspendedin methanol (40 ml). Glyoxylic acid monohydrate (3.32 g, 36.1 mmol, 100mol %) was added and the reaction was stirred at room temperature for 2hours. Sodium borohydride (1.50 g) was added carefully, and the reactionmixture bubbled very vigorously. The reaction mixture was stirred atroom temperature for 1 hour, then NaBH₄ (0.66 g) was added, followed bythe addition of glacial acetic acid (6 ml). After 15 minutes, NaBH₄(1.08 g) was added, and the reaction was stirred at room temperature for15 hours. An additional quantity of NaBH₄ (1.66 g) was then added andthe reaction was stirred at room temperature for 3 hours before it wasquenched with HCl (1M, 200 ml). The mixture was then extracted withethyl acetate (2×150 ml). The organic layers were combined, washed withsaturated NaCl solution (100 ml), dried over Na₂SO₄, filtered, and thesolvent removed under reduced pressure to afford a yellow solid (9.03 g)having ESMS Negative ion mode M-H⁺ Calculated 224 Found 224.

Example 6

Synthesis of N^(β-Boc-)2-(4-Phenyl-3-thiosemicarbazidyl) acetic acid

2-(4-Phenyl-3-thiosemicarbazidyl)acetic acid (8.93 g, 37.9 mmol, 100 mol%) and (BOC)₂O (9.10 g) were dissolved in dioxane (100 ml). Sodiumcarbonate (1M, 50 ml) and water (50 ml) were added and the mixture wasstirred at room temperature for 5 hours. Sodium hydroxide (1M, 40 ml)and an additional quantity of (BOC)₂O (6.21 g) were added and thereaction was stirred overnight at room temperature. The reaction wasquenched with citric acid (1M) and extracted with ethyl acetate (2×100ml). The organic layers were combined, washed with saturated NaCl (50ml), dried over Na₂SO₄, and filtered. The filtrate was concentratedunder reduced pressure to afford a gummy solid (19 g). The crude solidwas suspended in ether and a white solid was collected by filtration.The solid was washed with ether (100 ml) to obtain the desired product(3.17 g) having ESMS MH⁺ calculated 326, found 326.

Example 7

Synthesis of N^(a)-(triphenylmethylsulfenyl)-N^(b)-(Boc) azaglycine

^(t)-Butylcarbazate was condensed with glyoxylic acid monohydrate inmethanol. This crude hydrazone was then reduced by catalytichydrogenation over 10% Pd/C. This product was then mixed with dioxaneand base and a dioxane solution of triphenylmethanesulfenylchloride wasadded dropwise. The desiredN^(a)-(triphenylmethylsulfenyl)-N^(b)-(Boc)azaglycine (25 g) wasobtained on work-up.

Example 8

Solid Phase Peptide Synthesis of Peptides Using Alloc and FmocProtecting Groups

Solid phase peptide synthesis was carried out on a 0.050 mmol scaleusing an Advanced ChemTech model 348 peptide synthesizer modified tooperate under nitrogen pressure in the same manner as the model 396. The9-fluorenylmethyloxycarbonyl (Fmoc) group was employed for nitrogenprotection and diisopropylcarbodiimide (DIC)/hydroxybenzotriazole (HOBT)were used to activate the carboxyl groups for coupling. A variety ofresins were used such as Rink, Pal, and TentaGel S RAM for C-terminalamides and Wang, 2-chlorotrityl, or TentaGel S PHB for C-terminal acids.The alloc groups were cleaved on the machine in the manual mode bywashing the resin bound peptide with dichloromethane (3×2 ml portions)and then mixing the resin with a solution (2 ml) containingtetrakistriphenylphosphine palladium [0] (10 mg), and acetic acid (0.1ml). Tributyltinhydride (0.3 ml) was then added and the mixture wasvortexed for one hour. The reaction cell was then emptied, the resin waswashed with dichloromethane (3×2 ml) and standard Fmoc synthesis wasthen resumed. The peptides were cleaved from the resin with a solutionof trifluoroacetic acid (TFA), anisole and ethane dithiol for 1 to 3hours in the ratio 23:3:1. The crude cleavage mixture was then pouredinto ether to precipitate the crude peptide which was then purified byreverse phase HPLC using a Waters Delta Pak, Prep Pak C-18 cartridgesystem eluted with an appropriate gradient of TFA (0.1%) in water and/orTFA (0.1%) in acetonitrile (90%) and water (10%). The fractionscontaining the desired purified peptides were collected and the volatilesolvents were removed under reduced pressure to obtain the aqueoussolutions of the peptides which were then lyophilized. Samples of thelyophilized products were then sent for electrospray (ESMS) or fast atombombardment (FABMS) to confirm that the observed mass of the productsmatched the calculated mass of the desired peptide.

The table below shows some of the peptide sequences (SEQ ID NOS: 1,3-17)Respectively synthesized by the methods described above.

Peptide HPLC^(a) MW^(b) <GHWSYGLRPG—NH₂ 6.1 1183 <GHYSLEWKPG—NH₂ 6.21227 <GHWSYK(MaGC)LRPG—NH₂ 6.3 1488 <GHYSLK(MaGC)WKPG—NH₂ 6.3 1460<GHWSYK(Ms-azoGC)LRPG—NH₂ 6.1 1503 <GHYSLK(PtacGC)WKPG—NH₂ 6.9 1536AcNal_(d)Cps_(d)W_(d)SRK_(d)(MaGC)LRPA_(d)—NH₂ 8.2 1668<GHYSYLK(PtacGDsp)WKPG—NH₂ 6.6 1519 <GHYSLK(azaGGC)WKPG—NH₂ 6.5 1474Nal_(d)Cps_(d)W_(d)SRK_(d)(PtacGC)WKPG—NH₂ 8.1 1701<GHWSYK_(d)(MaGC)LRPG—NH₂ 6.3 1488AcNal_(d)Cps_(d)W_(d)SRK_(d)(AzaGPC)LRPA_(d)—NH₂AcNal_(d)Cps_(d)W_(d)SRK_(d)(MaPC)LRPA_(d)—NH₂AcNal_(d)Cps_(d)W_(d)SRK_(d)(PtacGC)LRPA_(d)—NH₂ <GHWSYK(iDGDep)LRPG—NH₂<GHWSYK(iECG)LRPG—NH₂ ^(a)HPLC Method [retention time in minutes]Solvent A is 0.1% trifluoroacetic acid in water, Solvent B is 0.1%trifluoroacetic acid in 90:10 acetonitrile/water Solvent flow rate is 3ml/min for 10 min then 5 ml/min for 5 min Gradient is 0 to 100% B over10 min then 100% B for 5 min ^(b)Electrospray mass spectrum values (MH⁺)Abbreviations used in Table: <G: pyroglutamic acid PtacG:2-(4-phenyl-3-thiosemicarbazidyl)acetic acid or PhNHCSNHNHCH₂CO2₂H Ma:mercaptoacetic acid azaG: azaglycine or H₂NNHCH₂CO₂H Dap:2,3-diaminoproprionic acid Nal: 2-naphthylalanine Cpa:4-chlorophenylalanine K_(d): the subscript d denotes that the D isomerwas used K(MaGC): the parentheses denote that enclosed amino acids areattached to the e amine of lysine and the first amino acid attached is Cfollowed by G and ending in Ma iD: isoaspartic acid iE: isoglutamic acid

Example 9

Radiolabeling with TC-99m

A Glucoscan (DuPont) vial was reconstituted with 2.18 mCi of NaTcO₄ in 1ml saline to form the Tc-99m-gluceptate complex. <GHWSYK(MaGC)LRPG amide(SEQ ID NO:4) (IMP3) was prepared as above. Tc-99m-IMP3 was prepared bymixing 360 μl (874 uCi) of Tc-99m-gluceptate with 640 μl of peptide insaline. The initially formed precipitate disappeared upon heating for 15min at 75° C. An instant TLC(ITLC) strip developed in H₂O:EtOH:NH₄OHmixture (5:2:1) showed 6.2% of the activity at the origin as colloids.HPLC showed 100% of the activity bound to the peptide with a RT of 6.95min, whereas the unlabeled peptide eluted at 6.4 min under the same HPLCconditions (reversed phase C-18 column, gradient of 0-100% B in 10 minat a flow rate of 3 ml/min, where A is 0.1% TFA in H₂O and B is 90%CH₃CN, 0.1% TFA). Recovery from the HPLC column was 85% of the injectedactivity.

IMP3 was formulated and lyophilized for Tc-99m labeling in the amountsshown below:

IMP3 (μg) Sn (μg) αDG/Sn 1. 250 23 14 2. 100 23 14 3. 250 15 14

The lyophilized vials were reconstituted with ˜900 μuCi of NaTcO₄ insaline. Cloudiness was observed in all the vials. The vials were heatedfor 15 min at 75° C., but turbidity persisted. ITLC analysis forcolloids showed 14, 21 and 9% colloids at the origin for vials 1, 2, and3, respectively.

In order to prevent the precipitation during Tc-99m labeling.α-D-glucoheptonate(aDG) and tartrate ratios to Sn(II) were varied in thelyophilized vials. The following vials were formulated and lyophilized(250 μg of IMP3 with 25 μg Sn(II)) with tartrate and αDG ratios as shownbelow. The vials were reconstituted with ˜500 μCi of NaTcO₄ in 1 mlsaline. Observatoins are indicated in the observation column. ITLCstrips were developed after 15 min at room temperature following heatingat 75° C. for 15 min.

tartrate/Sn pH Observation colloid, RT colloid, 75° C. 1. 50 5.3 ppt 2.100 5.3 ppt 3. 500 5.3 ppt clears 17% 2.4% upon mixing αDG/Sn pHObservation colloid, RT colloid, 75° C. 4. 25 5.3 ppt 5. 50 5.3 ppt 6.100 5.3 turbid 7. 500 5.3 slight turbidity 25% 3.5% 8. 1000 5.3 clear3.3%  3.1%

The protocol above was repeated for vials 3, 7 and 8 and colloids weredetermined to be 5.3, 3.8, and 4.6%, respectively after heating 15 minat 75° C. A single broad peak was observed on a reversed HPLC column ata RT of 7 min. Results from labeling other peptides with technetium-99are shown in the table below: (SEQ ID NO:4-8, 12, 9-11 respectively)

HPLC retention HPLC retention time Electrospray Peptide time (UV)(radiometric) mass spectrum <GHWSYK(MaGC)LRPG.amide 6.35 6.90 1488<GHYSLK(MaGC)WKPG.amide 6.48 7.07 1460 <GHWSYK(Ma-azaGC)LRPG.amide 6.557.02 1503 <GHYSLK(Ptac-GC)WKPG amide 7.05 7.60 1536AcNal_(d)Cps_(d)W_(d)SRK_(d)(MaGC)LRPA_(d)—NH₂ 8.50 (27%), 9.00 (68%)<GHWSYK_(d)(MaGC)LRPG—NH₂ 6.83 (95%) <GHYSYLK(PtacGDap)WKPG—NH₂ 7.06(96%) <GHYSLK(azaGGC)WKPG—NH₂ 6.60 (100%)Nal_(d)Cpa_(d)W_(d)SRK_(d)WKPG—NH₂ 8.43 (97%)

Abbreviations used in the table are the same as in Example 8 supra.

Example 10

Radiolabeling of IMP-3 Re-188

IMP3, (<GHWSYK(MaGC)LRPG amide) (SEQ ID NO:4) was synthesized as above.IMP 3 has a retention time of 6.4 min on a reversed phase C-18 columnusing a gradient of 0-100% B in 10 min at a flow rate of 3 ml/min whereA is 0.1% TFA in H₂O and B is 90% CH₃CN, 0.1% TFA.

IMP3 was formulated in 1 mg and 250 μg amounts with 450 μg Sn(II) andα-D-glucoheptonate at a ratio of 1:17.5, and lyophilized. Thelyophilized vials of IMP3 (1 mg and 250 μg) were reconstituted with 617and 578 μCi of NaReO₄ in saline. The vials were heated for 15 min at 75°C. HPLC analysis under the conditions described above showed singlepeaks at RT of 7.0 min for both vials. The effluent was collected andcounted on a γ-counter. For the 1 mg vial, the recovery of activity was88% whereas the recovery was 77% for the 250 μg vial. Colloid analyseson an ITLC strip developed in H₂O:EtOH:NH₄OH(5:2:1) showed 1.4 and 1.2%of the activity at the origin for 1 mg and 250 μg vials, respectively.

Re-188 labeling at room temperature did not proceed as well as at 75° C.At room temperature, only a few percent of the activity (<5%) wasincorporated into the peptide and the rest of the activity eluted in thevoid volume (1.2 min).

Example 11

In vitro Receptor Binding Assays

The human breast adenocarcinoma cell lines MCF-7, SK-BR-3, andMDA-MB-231 were purchased from the American Type Culture Collection,Rockville, Md. Cells were grown in DMEM supplemented with 5% fetalbovine serum. 5% defined equine serum, penicillin (100 U/ml),streptomycin (100 μg/ml), and L-glutamine (2 mM). The cells wereroutinely passaged after detachment with trypsin and 0.2% EDTA.

Specificity of the unlabeled peptides is determined by competitive cellbinding assay. Target cells are washed with fresh medium, and adjustedto 5×10⁵ cell/ml. 100 μl of the cell suspension (100 μl) is added perwell to a 96-well microtiter plate. The cells are allowed to attach andare then treated with different concentrations of the peptides in thepresence of ¹²⁵I-LHRH (Amersham Life Science, Arlington Heights, Ill.2,000 Ci/mmol). Following a 2 h incubation at room temperature withshaking, the cells are washed twice and the radioactivity associatedwith the cells is counted and the concentration of the peptides thatcause 50% inhibition on the binding of the labeled LH-RH is compared.

To determine receptor binding constants, serial dilutions ofradiolabeled LHRH are incubated with 5×10⁵ cells in a 96-well plate. Allassay are performed in triplicates both with or without a highconcentration of unlabeled LHRH to allow determination of specificallybound peptide. After a 2 h incubation at room temperature, the cells arewashed and counted. The equilibrium association constant, K_(a), and thetotal number of receptor sites per cell are determined by Scatchardanalysis.

Example 12

Biodistribution Studies

MCF-7 tumor cells are injected into estrogen-dosed nude mice, and atumor is allowed to develop. Carrier-free ¹²⁵I-LHRH (˜2000 Ci/mmol,Amersham Life Science) is injected into the mice and the mice aresacrificed at 5 min, 30 min, 1 hr, and 3 hr (3 animals per time point).The major organs, as well as the blood, and the tumor are removed,weighed, and counted to determine the percent injected dose per gram (%ID/g) in each organ.

Increasing amounts of unlabeled LHRH in 5 doses (from 0 to 0.1 mg) arethen mixed with the ¹²⁵I-LHRH and injected into the tumor-bearing nudemice (3 animals/dose) which are then sacrificed at time pointsdetermined from the previous experiment. This then allows determinationof the LHRH receptor capacity in the nude mouse model.

Tc-99m labeled peptides that demonstrate superior in vitro receptoraffinity as determined above are then screened in the MCF-7 nude mousemodel. The Tc-99m labeled peptide is purified by HPLC to obtain thepeptide carrier free metal complex for these studies if the LHRHreceptor capacity is too low to tolerate the presence of excess peptide.The biodistribution of the Tc-99m label (3 animals per peptide) ismonitored on a gamma camera equipped with a pinhole collimator. In theinitial screen the animals are sacrificed after 4 hr and thebiodistribution determined as described above. In subsequent experimentsthose Tc-99m labeled peptides that provide clear tumor images in theexperiment described above are screened in additional animals (3 pertime point), sacrificing at 15 min, 1 hr and 3 hr. The peptides are alsotested in a blocking assay using LHRH. Coinjection of LHRH decreasestumor uptake of the radiolabeled peptides in a dose-dependent manner,demonstrating that the in vivo tumor uptake is specific.

The invention has been disclosed broadly and illustrated in reference torepresentative embodiments described above. Those skilled in the artwill recognize that various modifications can be made to the presentinvention without departing from the spirit and scope thereof.

The invention has been disclosed broadly and illustrated in reference torepresentative embodiments described above. Those skilled in the artwill recognize that various modifications can be made to the presentinvention without departing from the spirit and scope thereof.

What is claimed is:
 1. A peptide comprising the amino acid sequenceX¹-X²-X³-ser-X⁴-X⁵-X⁶-X⁷-pro-X⁸-NH₂, wherein X¹ is pyroglutamic acid orD-acetylnaphthylalanine, X² is histidine or D-4-chlorophenylalanine, X³is D- or L-tryptophan or tyrosine, X⁴ is tyrosine, leucine, or arginine,X⁶ is leucine or tryptophan, X⁷ is arginine or lysine, X⁸-NH₂ is glycineamide or D-alanine amide, and wherein X⁵ is an amino acid derivativecapable of stably chelating technetium-99m, rhenium-186, or rhenium-188,and has the structure:

wherein R¹ is H, OH, peptide, a sugar, a targeting molecule, loweralkyl, substituted lower alkyl, or a protecting group that can beremoved under the conditions of peptide synthesis; R² is H, lower alkyl,or substituted lower alkyl; W is from 1-20 atoms long and is selectedfrom the group consisting of cycloalkyl, aryl, or alkaryl groups, asubstituted or unsubstituted alkylene chain, and a chain substitutedwith at least one heteroatom; Z is an amino acid or a peptide containing2-5 residues, or Z is COCH₂ or COCH(CH₂SP²), in which P² is H or asulfur protecting group; A and D are the same or different and each isselected from the group consisting of H, COCH₂NR³NR⁴C(S)NHR⁵,COCH₂NR⁶NR⁷C(S)NHR⁸, COCH₂NR⁹NR¹⁰C(O)CH₂SP², CONR¹¹NR¹²C(O)CH₂SP²,NR¹³C(S)NHR¹⁴, or COCH₂NR¹⁵COCH₂SP²; R³, R⁴, R⁶, R⁷, R⁹, R¹⁰, R¹¹, R¹²,R¹³, and R¹⁵ are the same or different, and each represents H, loweralkyl, or substituted lower alkyl; and R⁵, R⁸, and R¹⁴ are the same ordifferent and each is H, lower alkyl, substituted lower alkyl, aryl, orsubstituted aryl; or X⁵ is selected from the group consisting of:

wherein said amino acid sequence contains at least one thiol orthiocarbonyl group.
 2. A peptide according to claim 1, wherein X⁵ isselected from the group consisting of:


3. A peptide according to claim 2 1, wherein X¹ is pyro-glutamic acid,X² is histidine, and X⁸ is glycine amide.
 4. A peptide according toclaim 3, wherein X³ is tyrosine, X⁴ is leucine, X⁶ is tryptophan, and X⁷is lysine.
 5. A peptide according to claim 4, wherein X⁵ is


6. A peptide according to claim 5 4, wherein X⁵ is


7. A peptide according to claim 4, wherein X⁵ is


8. A peptide according to claim 3, wherein X³ is tryptophan, X⁴ istyrosine, X⁶ is leucine, and X⁷ is arginine.
 9. A peptide according toclaim 8, wherein X⁵ is


10. A peptide according to claim 8, wherein X⁵ is


11. A peptide according to claim 8, wherein X⁵ is


12. A peptide according to claim 2, wherein X¹ isD-acetylnaphthylalanine, X² is D-4-chlorophenylalanine, X³ isD-tryptophan, X⁴ is arginine, X⁶ is leucine, X⁷ is arginine, and X⁸-NH₂is D-alanine amide.
 13. A peptide according to claim 12, wherein X⁵ is


14. A peptide according to claim 12 1, wherein X⁵ is


15. A peptide according to claim 12 1, wherein X⁵ is


16. A peptide according to claim 12, wherein X⁵ is


17. A peptide according to claim 2 1, wherein X¹ isD-acetylnaphthylalanine, X² is D-4-chlorophenylalanine, X³ isD-tryptophan, X⁴ is arginine, X⁶ is tryptophan, X⁷ is lysine, and X⁸-NH₂is glycine amide.
 18. A method of preparing a metal-chelatingcomposition, comprising contacting a solution of a peptide with stannousions, wherein said peptide comprises the amino acid sequenceX¹-X²-X³-ser-X⁴-X⁵-X⁶-X⁷-pro-X⁸-NH₂, wherein X¹ is pyroglutamic acid orD-acetylnaphthylalanine, X² is histidine or D-4-chlorophenylalanine, X³is D- or L-tryptophan or tyrosine, X⁴ is tyrosine, leucine, or arginine,X⁶ is leucine or tryptophan, X⁷ is arginine or lysine, X⁸-NH₂ is glycineamide or D-alanine amide, X⁵ is an amino acid derivative capable ofstably chelating technetium-99m, rhenium-186, or rhenium-188, and hasthe structure:

wherein R¹ is H, OH, a peptide, a sugar, a targeting molecule, loweralkyl, substituted lower alkyl, or a protecting group that can beremoved under the conditions of peptide synthesis; R² is H, lower alkyl,or substituted lower alkyl; W is from 1-20 atoms long and is selectedfrom the group consisting of cycloalkyl, aryl, or alkaryl groups, asubstituted or unsubstituted alkylene chain, and a chain substitutedwith at least one heteroatom; Z is an amino acid or a peptide containing2-5 residues, or Z is COCH₂ or COCH(CH₂SP²), in which P² is H or asulfur protecting group; A and D are the same or different, and each isselected from the group consisting of H, COCH₂NR³NR⁴C(S)NHR⁵,COCH₂NR⁶NR⁷C(S)NHR⁸, COCH₂NR⁹NR¹⁰C(O)CH₂SP², CONR¹¹NR¹²C(O)CH₂SP²,NR¹³C(S)NHR¹⁴, or COCH₂NR¹⁵COCH₂SP²; R³, R⁴, R⁶, R⁷, R⁹, R¹⁰, R¹¹, R¹²,R¹³, and R¹⁵ are the same or different, and each represents H, loweralkyl, or substituted lower alkyl; and R⁵, R⁸, and R¹⁴ are the same ordifferent and each is H, lower alkyl, substituted lower alkyl, aryl, orsubstituted aryl, or X⁵ is selected from the group consisting of:

wherein said amino acid sequence contains at least one thiol orthiocarbonyl group; and then contacting said solution with^(99m)pertechnetate, ¹⁸⁶perrhenate or ¹⁸⁸perrhenate and recovering theradiolabeled peptide.
 19. The method of claim 18, wherein said peptidespecifically binds cells or tissues that express LHRH receptors.
 20. Themethod of claim 18, wherein said radionuclide is selected from ¹⁸⁸Re- or¹⁸⁶Re-perrhenate and ⁹⁹Tc-pertechnetate.
 21. A method of imaging atumor, an infectious lesion, a myocardial infarction, a clot,artherosclerotic plaque, or a normal organ or tissue, comprisingadministering to a human patient a radiolabeled peptide thatspecifically binds to cells or tissues that express LHRH receptors,together with a pharmaceutically acceptable carrier, and, after asufficient time for said radiolabeled peptide to localize and fornon-target background to clear, the site or sites of accretion of saidradiolabeled peptide detecting by an external imaging camera, whereinsaid radiolabeled peptide is prepared by contacting a solution of apeptide with stannous ions, wherein said peptide comprises the aminoacid sequence X¹-X²-X³-ser-X⁴-X⁵-X⁶-X⁷-pro-X⁸-NH₂, wherein X¹ ispyroglutamic acid or D-acetylnaphthylalanine, X² is histidine orD-4-chlorophenylalanine, X³ is D- or L-tryptophan or tyrosine, X⁴ istyrosine, leucine, or arginine, X⁶ is leucine or tryptophan, X⁷ isarginine or lysine, and X⁸-NH₂ is glycine amide or D-alanine amide, X⁵is an amino acid derivative capable of stably chelating technetium-99m,rhenium-186, or rhenium-188, and has the structure:

wherein R¹ is H, OH, a peptide, a sugar, a targeting molecule, loweralkyl, substituted lower alkyl, or a protecting group that can beremoved under the conditions of peptide synthesis; R² is H, lower alkyl,or substituted lower alkyl; W is from 1-20 atoms long and is selectedfrom the group consisting of cycloalkyl, aryl, or alkaryl groups, asubstituted or unsubstituted alkylene chain, and a chain substitutedwith at least one heteroatom; Z is an amino acid or a peptide containing2-5 residues, or Z is COCH₂ or COCH(CH₂SP²), in which P² is H or asulfur protecting group; A and D are the same or different, and each isselected from the group consisting of H, COCH₂NR³NR⁴C(S)NHR⁵,COCH₂NR⁶NR⁷C(S)NHR⁸, COCH₂NR⁹NR¹⁰C(O)CH₂SP², CONR¹¹NR¹²C(O)CH₂SP²,NR¹³C(S)NHR¹⁴, or COCH₂NR¹⁵COCH₂SP², R³, R⁴, R⁶, R⁷, R⁹, R¹⁰, R¹¹, R¹²,R¹³, and R¹⁵ are the same or different, and each represents H, loweralkyl, or substituted lower alkyl; and R⁵, R⁸, and R¹⁴ are the same ordifferent and each is H, lower alkyl, substituted lower alkyl, aryl, orsubstituted aryl, or X⁵ is selected from the group consisting of:

wherein said amino acid sequence contains at least one thiol orthiocarbonyl group; and then contacting said solution with^(99m)pertechnetate, ¹⁸⁶perrhenate or ¹⁸⁸perrhenate and recovering theradiolabeled peptide.
 22. A method of preparing peptides according toclaim 1, comprising the coupling of amino acids and amino acid analoguesby solid phase peptide synthesis.